In previous research on this project we discovered the potent antiinflammatory activity of the neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH). alpha-MSH exerts marked antiinflammatory effects via its action in the periphery. In humans, we recently found alpha-MSH in sites of peripheral inflammation in which cytokines were also concentrated, which suggests local production and action of this antiinflammatory molecule. However, little is known about the mechanism(s) of action of alpha-MSH; we have described it as an "anticytokine" influence. Our recent observations on macrophages and neutrophils suggest that alpha-MSH is a significant factor in a series of elements/events that constitute autocrine/paracrine pathways regulating inflammatory activity. Tests of this hypothesis require examination of the influence of alpha-MSH relative to these elements/events, both separately and when considered together in the hypothetical pathways. with regard to one principal element, a major aim in the project is to verify the observation that alpha-MSH alters expression of the gene for nitric oxide (NO) synthase. Positive evidence would indicate that a single action of the peptide, inhibition of synthesis of cytostatic cytotoxic NO, can account for antagonism by alpha-MSH of effects of multiple proinflammatory cytokines. In tests of another aspect of the pathway, we will determine if our initial observations of an inhibitory effect of alpha-MSH on cytokine production by inflammatory cells are correct and if this influence of the peptide occurs via inhibition of expression of genes for the proinflammatory cytokines. Because of the great importance of cytokines in inflammation, results of these experiments on IL-1, TNFalpha, and lL-8 may reveal a significant new mechanism of antiinflammatory action of the peptide. Judging from previous knowledge of cell signals induced by alpha- MSH, the peptide may exert its effects on inflammatory cells by increasing intracellular cAMP. This hypothesis, and the importance of this second messenger to autocrine/paracrine actions of the peptide, will be explored in the research program. Experiments utilizing PCR techniques should indicate if mRNA for specific subtypes of recently cloned alpha-MSH receptors is present in inflammatory cells. Our preliminary studies with macrophages and neutrophils indicate that these inflammatory cells do have such receptors. The planned experiments should disclose the types of stimuli that induce certain receptors and cellular factors (NO, cytokines, cAMP) likely to be important to receptor regulation. Other experiments on macrophages and neutrophils are designed to disclose if cytokines induce expression of the gene for the proopiomelanocortin precursor of alpha-MSH, and production of the peptide itself and to reveal any influence of macrophage-derived alpha-MSH on neutrophil function. The results of these experiments, considered separately and collectively, will answer specific questions about the mechanisms of antiinflammatory action of alpha-MSH and its potential physiological role in autocrine/paracrine control of inflammatory reactions.